Efficient synthesis of double dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assay.
A fast cleaving non-nucleosidic tetramethylrhodamine dye-labeled support has been developed for automated synthesis of double dye-labeled oligodeoxyribonucleotides in high yield. A mixture (1:1:2) of t-butylamine:methanol:water is used for cleavage and deprotection of dye-labeled oligodeoxyribonucleotides without any degradation or modification of dyes and nucleobases. The cleavage rate of oligodeoxyribonucleotides is significantly increased by using a diglycolate ester linkage instead of the commonly used succinate linkage. These double dye-labeled probes are used in PCR for real time detection of a specific PCR product. Using a 5'-exonuclease assay, detected on the ABI PRISM 7700 Sequence Detection System, there was no distinguishable difference in performance of probes synthesized using the dye-labeled support compared with traditional post-synthetic attachment of rhodamine.
Mullah
B
B
PE Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA. mullahbk@perkin-elmer.com
Livak
K
K
Andrus
A
A
Kenney
P
P
D001483:Base Sequence; D004396:Coloring Agents; D005069:Evaluation Studies as Topic; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D015345:Oligonucleotide Probes / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012235:Rhodamines
D004396:Coloring Agents; D015345:Oligonucleotide Probes; D012235:Rhodamines; C005358:tetramethylrhodamine